Abstract: Hedgehog (Hh) signaling plays critical roles in pattern formation and cell growth control and is also involved in metabolic control. Further, aberrant Hh signaling causes diverse types of cancers. Transduction of the Hh signal requires the G protein-coupled receptor (GPCR) family protein Smoothened (Smo) in both insects and mammals. While many components in the Hh pathway have been identified, how the Hh signal is transduced through the 12-span transmembrane protein Patched (Ptc) to Smo is still unclear. This team has discovered 1) that Smo undergoes phosphorylation by multiple kinases, which leads to Smo cell surface accumulation and signaling activity; 2) that sumoylation induced by Hh promotes Smo activation; and 3) that ubiquitination downregulates Smo cell surface accumulation by promoting Smo endocytosis. Results from these studies suggest that, in response to Hh stimulation, multiple steps occur in Smo regulation. However, despite significant progress, key questions persist regarding the mechanism controlling Smo protein expression and activation: 1) How is Smo transcription specifically regulated? 2) Whether and how does Hh regulate lipolysis? 3) How does cholesterol activate Smo? and 4) Whether and how is the sodium pump involved in Ptc and Smo regulation? Recent discoveries by this group that Smo transcription is specifically regulated in adipose tissue, that an alternative cholesterol biosynthesis pathway regulates Smo accumulation and activation, and that the sodium pump plays a positive role in activating Smo, likely by regulating cholesterol on the plasma membrane are critical foundations to answering those questions. The overarching goal of this research program is to understand how Hh signals are sensed and transmitted to control downstream biological events that ultimately govern cell growth and patterning. Drosophila fat body and oenocyte have emerged as attractive models to study lipid metabolism and circulation. Published and preliminary findings in these models have indicated that lipid accumulation is regulated by highly conserved signaling pathways, and that Hh signaling controls not only lipogenesis but also lipolysis by regulating specific genes. These studies provide new tools and hypotheses for investigating the mechanisms of Smo signaling and the role of Hh/Smo signaling in regulating lipid metabolism, which particular relevance to such cancers as basal cell carcinoma and medulloblastoma. The proposed five- year program of studies uses a combination of genetic and biochemical approaches to build on prior contributions and to transition to newly emergent avenues of inquiry. The knowledge gained from this study will provide novel insights into mechanisms surrounding Smo suppression by Ptc and activation by cholesterol. In addition, expected outcomes will provide novel insights into human developmental disorders and promote development of diagnostic tools and novel therapeutic approaches to oppose Smo drug resistance using metabolism as a platform to transform current concepts of Hh signaling in cancer, obesity, and metabolic diseases.